The initial biochemical step in the mechanism of action of Epidermal Growth Factor (EGF), and polypeptide hormones in general, is reasoned to be the binding of the molecule to specific receptors on the cell surface. Our main objective, therefore, will be to investigate EGF-receptor interactions in a defined experimental system. This will require the use of cell free systems in which EGF forms a hormone receptor complex which initiates a defined biochemical reaction, so that binding can be coupled to biologic responses. We have chosen as our model the human epidermoid carcinoma cell line, A-431 because it has an extraordinarily high concentration of specific membrane receptors for EGF (2-3 million/cell). In our initial studies we have found that membrane preparations from the A-431 cells retain the ability to bind 125I-labeled EGF in a specific manner and that the binding of EGF to these membranes in vitro produced a marked stimulation of the phosphorylation of both endogenous and exogenous protein substrates in the presence of (lambda-32P) ATP. We propose to study: 1) the nature of the phosphorylation mechanisms sensitive to EGF in vitro and 2) to determine which membrane components are involved. We will determine if the membrane phosphorylation is due to stimulation of membrane associated protein kinases and the role of protein phosphatases, ATPases and the cyclic nucleotide system in this EGF sensitive in vitro model. Using gel electrophoresis and autoradiographic methods the specific membrane components will be identified and their topographical relationships to the membrane determined. The hormonal specificity and the tissue specificity of the EGF-sensitive phosphorylation will also be examined. The phosphorylated membrane components and the EGF-sensitive phosphorylation system will be characterized by solubilization, purification and reconstitution procedures. These studies will help to determine if the phosphorylation of membrane or membrane associated components is an initial event in the generation of intracellular signals by EGF to regulate cell growth and/or differentiation.